Method for producing cellulolytic and/or hemicellulolytic enzymes

ABSTRACT

The process for the production of cellulolytic and/or hemicellulolytic enzymes by a cellulolytic and/or hemicellulolytic microorganism according to the present invention comprises at least one phase for growth in the presence of a source of carbon and at least one phase for production in the presence of an inducing substrate, in which said inducing substrate is a mixture comprising 40% to 65% by weight of glucose or cellulosic hydrolysates, 21% to 25% by weight of lactose and 10% to 39% by weight of xylose or a solution of a lignocellulosic hemicellulosic hydrolysate, the sum of these three constituents being equal to 100%.

FIELD OF THE INVENTION

The invention relates to a process for the production of enzymes for thehydrolysis of lignocellulosic biomass.

PRIOR ART

The increase in bioethanol production capacity for its qualities for inbiofuels is a current “hot topic”. Incorporation targets are underdiscussion within the European Union, based on an initial proposition of20% use of renewable energy by 2020 with an incorporation of 10% ofbiofuels, subject to sustainability criteria which should favour secondgeneration biofuels produced from lignocellulosic biomass.

Lignocellulosic biomass is characterized by a complex structureconstituted by three principal polymers: cellulose, hemicellulose andlignin.

Conventionally, the process for the transformation of biomass intoethanol comprises a number of steps. A pre-treatment can rendercellulose and possibly hemicelluloses, which are the targets forenzymatic hydrolysis, accessible to enzymes. The pre-treatment is aimedat modifying the physical and physico-chemical properties of thelignocellulosic material with a view to improving accessibility to thecellulose trapped in the matrix of lignin and hemicellulose. Theenzymatic hydrolysis step can be used to transform cellulose andhemicelluloses into sugars using cellulolytic and/or hemicellulolyticenzymes.

The sugars obtained by hydrolysis of lignocellulosic biomass arepentoses (principally xylose and arabinose), disaccharides (cellobiose)and glucose, which can be fermented by microorganisms. Glucose may, forexample, be readily transformed into ethanol by the yeast Saccharomycescerevisiae during the alcoholic fermentation step.

Finally, a distillation step can separate and recover the productobtained from the fermentation, i.e. ethanol in the above case, from thefermentation must.

Various technico-economic studies have demonstrated the necessity ofreducing the costs linked to the enzymatic hydrolysis step in order tobring the cost of the ethanol produced to values close to that for theethanol obtained from starch.

Currently, industrial cellulases are principally produced by afilamentous fungus, Trichoderma reesei, because of its highcellulase-secreting power.

One of the ways of reducing costs consists of optimizing the operatingconditions for the cellulase production process in order to increase itsproductivity or to obtain an enzymatic cocktail with an improvedspecific activity.

In the presence of an inducing substrate, wild type strains ofTrichoderma reesei have the ability to secrete an enzymatic complexwhich is well suited to cellulose hydrolysis. The enzymes of theenzymatic complex contain three major types of activity: endoglucanases,exoglucanases and cellobiases. Other proteins, such as xylanases, whichare necessary for hydrolysis of the lignocellulosic biomass, are alsoproduced by Trichoderma reesei. The presence of an inducing substrate isindispensable to the expression of cellulolytic and/or hemicellulolyticenzymes.

Regulation of the genes for cellulases on various carbon sources hasbeen studied in detail. Glucose exerts a catabolic repression effect onthe production of cellulases. This is induced in the presence ofcellulose, its hydrolysis products such as cellobiose or certainoligosaccharides, in particular disaccharides such as lactose orsophorose (Ilmén et al. 1997, Appl. Environ. Microbiol. Vol 63, p1298-1306). The nature of the carbonaceous substrate has a majorinfluence on the composition of the enzymatic complex. Thus, xylose,associated with a carbonaceous inducing substrate such as cellulose orlactose, can be used to significantly improve the “xylanase activity”when it is present in limiting concentrations of the order of 0.5 to 1mM (Mach-Aigner et al., 2010 Applied and Environmental Microbiology, Vol76 N °6, p 1770-1776). Dissolving residual hemicelluloses (xylanes) withxylanases could promote enzymatic hydrolysis (Vàrnai et al., 2010 Enzymeand Microbial Technology Vol 46 p 185-193).

In order to obtain good enzyme productivities, it is necessary toprovide a source of carbon that can be rapidly assimilated in order toallow rapid growth of Trichoderma reesei, and an inducing substrate thatcan allow the expression of cellulases and secretion into the culturemedium. Cellulose can play a dual role. However, it is difficult to useon an industrial scale and it has been replaced by soluble carbonsources, such as lactose, which also act as an inducing substrate.

Other sugars such as cellobiose or sophorose have also been described asinducers (Ilmen et al. (1997), Foreman et al. (2003) Biol Chem 278, p31988-31997, Pakula et al. (2005) Microbiology, 151, p 135-143), but aretoo expensive to be used on an industrial scale.

In general, it has been observed that the production of cellulases byTrichoderma reesei with soluble substrates is much lower than thatobtained on cellulose in a batch mode (i.e. in a sealed medium) becauseof the repressor effect of sugars that are readily assimilated at highconcentrations.

Patent FR-B-2 555 603 in the name of the Applicant proposes thecontinuous supply of carbonaceous substrates in order to lift thecatabolic repression by limiting the residual concentration in thecultures and by optimizing the quantity of sugars in order to obtain abetter yield and better enzymatic productivity. The process described inthis patent proposes starting to supply the substrate using solublesugars as the source of carbon, optionally in the form of a mixture.However, a continuous supply limiting glucose or xylose not associatedwith lactose or with another cellulase inducer (sophorose, cellobiose,etc) cannot be used to obtain high enzyme productivity.

In industrial cellulolytic enzyme production processes, lactose remainsone of the most suitable substrates. In addition to being high, itsprice fluctuates a great deal and represents approximately one third ofthe cost price of the enzymes.

One of the solutions envisaged and presented in patent EP-B-0 448 430consists of using carbonaceous substrates obtained from a die, forexample hydrolysed hemicelluloses, as a source of inducing carbon.However, the productivity remains low, at about 50% compared with theprocess using lactose alone as an inducing substrate.

Patent application WO 2009/026716 describes a process for the productionof cellulases in which the hemicellulolytic derivatives represent morethan 40% of the carbon source and the sugars inducing cellulaseproduction represent in the range 3% to 20% of this mixture. Thatprocess can be used to produce at least twice the quantity of cellulasescompared with a process using only sugars obtained from hemicelluloses.However, the cellulase production performance is still lower than aprocess using only lactose.

Current research shows that, in order to improve cellulolytic and/orhemicellulolytic enzyme production processes, it is necessary to developnovel inducing substrates which perform at least as well as those usinglactose, and which cost less.

This constitutes the context of the present invention.

SUMMARY OF THE INVENTION

The present invention describes a process for the production ofcellulolytic and/or hemicellulolytic enzymes in which the inducingsubstrate is a mixture of glucose, lactose and xylose.

DETAILED DESCRIPTION OF THE INVENTION

The process for the production of cellulolytic and/or hemicellulolyticenzymes by a cellulolytic and/or hemicellulolytic microorganismaccording to the present invention comprises at least one phase forgrowth in the presence of a source of carbon and at least one phase forproduction in the presence of an inducing substrate, in which saidinducing substrate is a mixture of glucose or cellulosic hydrolysates,lactose and xylose or a solution of hemicellulolytic hydrolysates, thequantities of each of the constituents of the mixture being defined bythe following limits:

-   -   40% to 65% by weight of glucose or cellulosic hydrolysates;    -   21% to 25% by weight of lactose; and    -   10% to 39% by weight of xylose or a solution of hemicellulosic        hydrolysates;        the sum of these three constituents being equal to 100%.

The inducing substrate is free of any sugar other than the constituentslisted above. Thus, the relative proportions of each of the constituentsare selected such that the sum of the quantities by weight is equal to100%.

Thanks to the process of the invention using a mixture of particularsugars as an inducing substrate, the final concentrations of proteinsobtained are 50% to 60% higher than those obtained with lactose used asthe sole production substrate. They are also approximately 3 timeshigher than that obtained with improved mixtures which are rich inxyloses as described in the prior art document WO 2009/026716.

The process of the present invention uses a mixture based on glucose.More than 60% of lactose, an inducer of cellulase production, can bereplaced by glucose which, however, is known to be a repressor ofcellulase production, with improved final performance of the inducingmixture. Glucose is much cheaper than lactose and its solubility inwater is approximately 3 times higher. Thus, it is possible to reducethe volumes employed. Thus, in this process, glucose is not only used inthe growth phase, but also in the production phase in an amount of 60%of the mixture constituting the inducing substrate.

The glucose used in the mixture of sugars may also be replaced byglucose obtained from the cellulose enzymatic hydrolysis step, i.e.obtained directly from the process for the transformation oflignocellulosic biomass into ethanol. This contributes to reducing thecost of enzyme production by using co-products from the process. Thus,they are termed cellulosic hydrolysates.

The xylose used in the mixture constituting the inducing sugar may bereplaced by a solution of hemicellulolytic hydrolysates obtained fromthe process for the transformation of lignocellulosic biomass intoethanol, and in particular obtained from pre-treatment of the biomass.

Furthermore, the possibility of using a very highly concentrated mixtureof sugars can advantageously limit the risk of contamination.

The fact that xylose is added to the mixture constituting the inducingsubstrate means that the xylanase activity of the final enzymaticcocktail is greatly increased. The xylose may advantageously be replacedby a hemicellulosic hydrolysate solution obtained from the hydrolysis ofhemicelluloses during pre-treatment of the lignocellulosic biomass insecond generation biofuel production processes; it may be concentratedif necessary.

Preferably, the mixture constituting the inducing substrate comprises50% to 65% by weight of glucose or cellulosic hydrolysates, 22% to 24%by weight of lactose and 15% to 25% by weight of xylose or a solution ofhemicellulosic hydrolysates, optionally obtained from a pre-treatment ofthe lignocellulosic biomass, the sum of the constituents being equal to100%.

Highly preferably, the inducing substrate is a mixture constituted by60% by weight of glucose or cellulosic hydrolysates, 23% by weight oflactose and 17% by weight of xylose or hemicellulosic hydrolysates.

The carbonaceous substrate used during the growth phase is selected fromglucose, xylose, lactose, residues obtained after ethanolic fermentationof monomeric sugars of the enzymatic hydrolysates of cellulosic biomassand/or an unrefined extract of hydrosoluble pentoses possibly derivingfrom the pre-treatment of a cellulosic biomass.

Highly preferably, the growth substrate is glucose.

The industrial strains used belong to the species Trichoderma reesei,modified in order to improve the cellulolytic and/or hemicellulolyticenzymes by mutation-selection processes. An example which can be citedis the strain IFP CL847. Strains improved by genetic recombinationtechniques may also be used. They have to be deleted for catabolicrepression by glucose (Δ CRE1); an example is CL847.

These strains are cultivated in agitated, aerated bioreactors underconditions which are compatible with their growth and the production ofenzymes. The conditions are such that the pH is adjusted to between 3.5and 6 and the temperature is in the range 20° C. to 35° C. Preferably, apH of 4.8 and a temperature of 27° C. are selected during the growthphase and a pH of 4 and a temperature of 25° C. are selected during theproduction phase. The degree of aeration, expressed as the volume of airper volume of reaction medium per minute, or vvm, applied during theprocess is in the range 0.3 to 1.5 min⁻¹, and the rate of rotation, rpm,must allow the O₂ pressure to be adjusted to between 20% and 60%.Preferably, an aeration of 0.5 vvm and agitation to allow the O₂pressure to be adjusted to 30% are selected.

Depending on its nature, the carbonaceous substrate selected to obtainthe biomass is introduced into the bioreactor before sterilization, oris sterilized separately and introduced into the bioreactor aftersterilization thereof in order to produce an initial sugar concentrationof 15 to 60 g/L.

In view of the production phase, an aqueous solution containing theinducing substrate constituted by a glucose/lactose/xylose mixture orhemicellulose hydrolysate solution selected for the enzyme productionphase is prepared in a concentration of 350 to 600 g/L in the feedsolution used.

Preferably, the concentration is in the range 450 to 550 g/L.

The aqueous solution is injected after the initial substrate has beenexhausted in order to provide an optimized quantity. The flow rate atwhich the solution is supplied is 30 to 45 mg per gram of cells perhour.

The residual sugar concentration in the culture medium is less than 1g/L during the production phase (fed-batch) in order to limit theproduction of biomass. Preferably, this concentration is less than 0.5g/L, still more preferably less than 0.1 g/L.

EXAMPLES

In the examples which follow, Example 1 presents a culture using glucoseas the carbonaceous substrate during the growth phase and the productionphase. This example demonstrates the low production of proteins obtainedwhen this sugar is used as the sole carbonaceous substrate even if theflow is limiting during the fed-batch phase (residual concentration ofglucose close to 0). The second example represents the referencefermentation using lactose as the carbonaceous substrate during thegrowth phase and the production phase which allows high proteinproduction. Example 3 reproduces an experiment using a mixture in theproduction phase as described in patent application WO 2009/026716containing a high proportion of xylose allowing, according to theauthors, the cellulase production to be greatly increased compared withan experiment in which xylose or hemicellulosic derivatives are used asthe sole carbonaceous substrates. Examples 4 to 6 are those of thepatent, in accordance with the process of the present invention.

Example 1 Production of Enzymes on Glucose (Not in Accordance with theInvention)

The production of cellulases was carried out in a mechanically stirredbioreactor. The mineral medium had the following composition: KOH 1.66g./L, H₃PO₄ 85% 2 mL/L, (NH₄)₂SO₄ 2.8 g/L, MgSO₄.7 H₂O 0.6 g/L, CaCl₂0.6 g/L, MnSO₄ 3.2 mg/L, ZnSO_(4.)7 H₂O 2.8 mg/L, CoCl_(2.)10H₂O 4.0mg/L, FeSO_(4.)7 H₂O 10 mg/L, Corn Steep 1.2 g/L, anti-foaming agent 0.5mL/L.

The bioreactor containing the mineral medium was sterilized at 120° C.for 20 minutes; the glucose carbonaceous source was sterilized from 120°C. for 20 minutes then added to the bioreactor under sterile conditionsin order to produce a final concentration of 30 g/L. The bioreactor wasprimed to 10% (v/v) with the liquid pre-culture of the CL847 strain ofTrichoderma reesei. The mineral medium of the pre-culture was identicalto that of the bioreactor apart from adding potassium phthalate in aconcentration of 5 g/L to buffer the pH. Growth of the fungus inpre-culture was carried out using glucose as the carbonaceous substrateat a concentration of 30 g/L. Growth of the inoculum lasted 2 to 3 daysand was carried out at 28° C. in a shaker incubator. Transfer to thebioreactor was carried out if the residual concentration of glucose wasless than 15 g/L.

The experiment carried out in the bioreactor comprised two phases:

-   -   a phase for growth on a glucose carbonaceous substrate (initial        concentration=30 g/L) at a temperature of 27° C. and a pH of 4.8        (regulated with 5.5 M ammonia). Aeration was at 0.5 vvm and        agitation was increased between 200 and 800 rpm as a function of        the pO₂ (dissolved oxygen pressure), which was kept above 30%;    -   an enzyme production phase. When the initial substrate of the        fermenter was exhausted, the 250 g/L glucose solution was        injected continuously at a flow rate of 30 to 40 mg per gram of        cells per hour up to 164 hours. The temperature was reduced to        25° C. and the pH to 4 until the end of culture. The pH was        adjusted by adding a 5.5 N ammonium solution which provided the        nitrogen necessary for synthesis of the excreted proteins. The        dissolved oxygen content was kept above 15% to 20% by adjusting        the aeration and agitation.

Enzyme production was followed by assaying the extracellular proteinsusing the Lowry method and BSA standard, after separation from themycelium by filtering or centrifuging. The cellulolytic activities whichwere determined were:

-   -   the filter paper activity (FPU=filter paper unit), which meant        that the overall activity of the endoglucanase and exoglucanase        enzymatic pool could be assayed;    -   the aryl β-glucosidase and xylanase activities for the specific        activities.

The FPU activity was measured on Whatman No 1 paper (procedurerecommended by the IUPAC Biotechnology Commission) at an initialconcentration of 50 g/L; the test sample from the enzymatic solution tobe analysed which liberated the equivalent of 2 g/L of glucose(colorimetric assay) in 60 minutes was determined. The principle offilter paper activity is to determine, by DNS (dinitrosalicylic acid)assay, the quantity of reduced sugars obtained from a Whatman No 1 paper(procedure recommended by the IUPAC Biotechnology Commission).

The substrate used to determine the aryl β-glucosidase activity wasp-nitrophenyl-β-D-glucopyranoside (PNPG). It is cleaved by β-glucosidaseto liberate p-nitrophenol.

One aryl β-glucosidase activity unit is defined as the quantity ofenzyme necessary to produce 1 μmole of p-nitrophenol from PNPG perminute and is expressed in IU/ml. The principal of xylanase activityassay resides in determining, by DNS assay, the quantity of reducedsugars obtained from the hydrolysed xylanase solution. This assay methoduses the reducing properties of the sugars, principally xylose. Thexylanase activity is expressed in IU/ml, and corresponds to the quantityof enzyme necessary to produce 1 μmole of xylose per minute.

The specific activities were obtained by dividing the activitiesexpressed in IU/ml by the concentration of proteins. They are expressedin IU/mg.

The analytical determinations on the final must of Example 1 producedthe following results:

The analytical determinations of the final must produced the followingresults:

Biomass 15.2 g/L

Proteins 2.9 g/L

FPU 1.4 IU/mL

Xylanase 29.1 IU/mg

Specific β-glucosidase 0.35 IU/mg

The CL847 strain was presented as being derepressed to the catabolicrepression exerted by glucose on the production of cellulase (it isdeleted from the CRE1 gene). It appears that even for a fed-batchreaction carried out under glucose limitation conditions, the finalproduction of proteins is very low.

Example 2 Production of Enzymes on Lactose (Not in Accordance with theInvention)

Enzyme production was carried out under the same conditions as inExample 1. The carbonaceous substrate during the growth and productionphases was pure lactose. Lactose is an important inducer in cellulaseproduction. It is this substrate which is the most widely used for theproduction of cellulases on an industrial scale.

After 30 hours of growth, after exhausting the initial substrate, the250 g/L fed-batch solution was injected continuously at a flow rate of35 mg per gram of cells per hour up to 164 hours.

The analytical determinations of the final must produced the followingresults: Biomass 13.5 g/L

Proteins 37.8 g/L

FPU 22.1 IU/mL

Xylanase 408.5 IU/mg

Specific β-glucosidase 0.96 IU/mg

Example 3 Production with Mixture of 97% Xylose/3% Sugars Inducing theProduction of Cellulases CIC (Not in Accordance with the Invention)

The experiment was carried out under the same conditions as in Example1, using pure xylose as the sole carbonaceous substrate during thegrowth phase, and in the production phase the 97% xylose/3% CIC mixturepresented in patent application WO 2009/026716 A1. CICs (sugars inducingthe production of cellulases) are a mixture of sugars allowing theinduction of cellulase production. Its composition was as follows: 56%gentiobiose, 14% sophorose, 10% trehalose, 6% cellobiose, 6%maltotriose, 8% glucose.

After exhausting the initial substrate, the 360 g/L 97% xylose/3% CICmixture was injected, following the recommendations described, at a flowrate of 0.4 g of carbon/L/h.

The analytical determinations of the final must produced the followingresults:

Biomass 14.3 g/L

Proteins 18.7 g/L

FPU 6.4 IU/ml

Xylanase 6000.1 IU/mg

Specific FPU 0.34 IU/mg

Specific β-glucosidase 1.15 IU/mg

The final protein production was close to that presented in WO2009/026716 A1 where the authors obtained a final concentration ofproteins of 25 g/L with the P59G strain. The FPase specific activity(0.34 IU/mg) was low, while the xylanase activity was very high.

Example 4 Production with Lactose (23%)/Glucose (60%)/Xylose (17%)Mixture at 500 g/L (in Accordance with the Invention)

The experiment of Example 4 was carried out under the same conditions asin Example 1 with glucose as the carbonaceous substrate for growth in anamount of 60 g/L. During the fed-batch production phase, we then used asolution in which three carbonaceous substrates were mixed in thefollowing proportions: lactose (23%)/glucose (60%)/xylose (17%), at aconcentration of 500 g/L. This mixture was injected at 45 mg/g/h. Theanalytical determinations of the final must produced the followingresults:

Biomass 26.1 g/L

Proteins 61.8 g/L

FPU 33.7 IU/ml

Xylanase 4017 IU/mg

Specific FPU 0.55 IU/mg

Specific β-glucosidase 1.2 IU/mg

The final protein production was more than 3 times higher than that ofExample 3. The filter paper activity, which is an indicator of cellulaseactivity, was approximately 5 times higher.

The xylanase activity was 10 times higher than that of Example 2 carriedout with lactose as the sole growth and production carbonaceoussubstrate, but approximately 50% lower than that of Example 3.

Example 5 Use of Lactose (23%)/Glucose (60%)/C5 Sugar Solution (17%)Mixture, at 500 g/L (in Accordance with the Invention)

Example 5 was carried out under the same conditions as Example 4. Theglucose was used during the growth phase, at a concentration of 15 g/L.The xylose of the fed-batch solution was replaced by a solution ofsoluble hemicellulosic solution or C5 sugar solution obtained from awheat straw impregnated with 0.08 N sulphuric acid and pre-treated bysteam explosion (19 bar, 5 minutes). The mixture was injected at 30mg/g/h for 170 h.

The analytical determinations of the final must produced the followingresults:

Biomass 19.1 g/L

Proteins 57.3 g/L

FPU 25.1 IU/ml

Xylanase 1151 IU/mg

Specific FPU 0.44 IU/mg

Specific β-Glucosidase 1.4 IU/mg

This experiment allowed the protein production to be increased by morethan 50% compared with Example 2 and the xylanase activity was almost 3times better. The majority of lactose was replaced by glucose and C5sugar solution during the growth phase and production phase. Theconcentration of glucose during the growth phase was reduced comparedwith Example 4, by 60 to 15 g/L. The concentration of biomass wasreduced from 26 to 19 g/L but this had little impact on the finalconcentration of proteins (7% lower). Thus, use of the sugars wasoptimized and the cost of the carbon source was considerably reduced.

Example 6 Use of Lactose (23%)/C6 Hydrolysates (56%)/Xylose (21%)Mixture, 500 g/L (in Accordance with the Invention)

Example 6 was carried out under the same conditions as Example 4. Theglucose was used during the growth phase, at a concentration of 15 g/L.The glucose of the fed-batch solution was replaced by a solution ofhydrolysate obtained from the enzymatic hydrolysis of a wheat strawimpregnated with 0.08 N of sulphuric acid and pre-treated by steamexplosion (19 bar, 5 minutes). The enzymatic hydrolysis of the wheatstraw was carried out at 50° C. and at a pH of 4.8. It resulted inhydrolysis of 95% of the cellulose into glucose.

Lactose and xylose were dissolved in this solution so as to result in amixture:

Lactose (23%)/C6 hydrolysates (56%)/xylose (21%), at 500 g/L.

The mixture was injected at 30 mg/g/h for 170 h during the fed-batchphase.

The analytical determinations of the final must produced the followingresults:

Biomass 16.1 g/L

Proteins 43.1 g/L

FPU 31.4 IU/ml

Xylanase 6595.1 IU/mg

Specific FPU 0.73 IU/mg

Specific β-glucosidase 1.2 IU/mg

The enzymatic cocktail obtained was very high quality, since the finalFPase activity was close to the activity obtained in Example 4 even ifthe protein concentration was lower. The final FPase activity wasapproximately 5 times higher than that obtained with Example 3 andapproximately 50% higher than that obtained during Example 2 wherelactose was used as the sole carbonaceous substrate during the fed-batchphase.

1. A process for the production of cellulolytic and/or hemicellulolyticenzymes by a cellulolytic and/or hemicellulolytic microorganism,comprising at least one phase for growth in the presence of a source ofcarbon and at least one phase for production in the presence of aninducing substrate, in which said inducing substrate is a mixture ofglucose or cellulosic hydrolysates, lactose and xylose or a solution ofhemicellulolytic hydrolysates, the quantities of each of theconstituents of the mixture being defined by the following limits: 40%to 65% by weight of glucose or cellulosic hydrolysates; 21% to 25% byweight of lactose; and 10% to 39% by weight of xylose or a solution ofhemicellulosic hydrolysates; the sum of these three constituents beingequal to 100%.
 2. A process according to claim 1, in which themicroorganism belongs to the species Trichoderma reesei and is deletedfor catabolic repression by glucose.
 3. A process according to claim 1,in which the inducing substrate is supplied in solution, theconcentration of inducing substrate in the supply solution used duringthe production phase being 350 to 600 g/L.
 4. A process according toclaim 3, in which the concentration is in the range 450 to 550 g/L.
 5. Aprocess according to claim 1, in which the mixture constituting theinducing substrate comprises 50% to 65% by weight of glucose orcellulosic hydrolysates, 22% to 24% by weight of lactose and 15% to 25%by weight of xylose or a solution of hemicellulosic hydrolysates, thesum of the constituents being equal to 100%.
 6. A process according toclaim 5, in which the inducing substrate is a mixture constituted by 60%by weight of glucose or cellulosic hydrolysates, 23% by weight oflactose and 17% by weight of xylose or hemicellulosic hydrolysates.
 7. Aprocess according to claim 1, in which the carbonaceous substrate usedduring the growth phase is selected from glucose, xylose, lactose,residues obtained after ethanolic fermentation of monomeric sugars ofthe enzymatic hydrolysates of cellulosic biomass and/or an unrefinedextract of hydrosoluble pentoses possibly derived from the pre-treatmentof a cellulosic biomass.
 8. A process according to claim 1, in which thepH is adjusted to between 3.5 and 6 and the temperature is in the range20° C. to 35° C.
 9. A process according to claim 1, in which the flowrate for introduction of the supply solution is 30 to 45 mg per gram ofcells per hour.
 10. A process according to claim 1, in which theresidual concentration of sugar in the culture medium during theproduction phase is less than 1 g/L, preferably less than 0.5 g/L andhighly preferably less than 0.1 g/L.